Within the field of biological diagnostics, research and analytical procedures require the investigation of nucleic acids from biological specimens, such as whole blood or fractions thereof. Such in vitro procedures require as a first step, the isolation of the nucleic acids. For example, relatively pure samples of genomic DNA are required in order to perform tests for genetic diseases, and recombinant technology requires isolation of both the vector DNA and the DNA to be cloned. In the detection of infectious agents, such as bacteria and vitally infected cells, DNA diagnostic procedures generally require cell lysis followed by detection the released DNA.
Generally, DNA does not exist as a free molecule in a cell, but instead exists as a complex association of DNA, RNA and proteins. This is a consequence of the role of DNA as the carrier of genetic information, and its involvment with RNA and various proteins in that function.
Because of this complex association of DNA with other materials in a specimen, effective DNA extraction requires that: the DNA be released through disrupted cell walls and membranes, DNA-protein complexes be dissociated by denaturation or proteolysis, and DNA be separated from other macromolecules. Various means are used in the art to accomplish one or more of these results. Cell lysis can be accomplished, for example, by freeze-thawing, ultrasonic means, shearing and other mechanical techniques, or by treatment with enzymes, surfactants or chelating agents. Proteases and other hydrolyzing agents can be used to dissociate DNA from proteins. Residual proteins and other macromolecules can be extracted using various solvents, such as phenol or other alcohols.
Some DNA isolation techniques are described in, for example, E. P. Publications 145,356 (published Jun. 19, 1985), 240,191 (published Oct. 7, 1987), and 245,945 (published Nov. 19, 1987), all of which use an alcohol and an enzymatic protein decomposer in certain sequences of steps. Generally, these procedures are directed to the extraction of vital DNA and involve a number of complicated steps which must be carried out with precision in order to obtain all available DNA. Thus, many of the known processes are labor intensive, require the use of undesirable solvents and are not readily automated.
Isolation or extraction of the nucleic acid of interest is necessary to take advantage of recent developments for amplification and detection of nucleic acids using polymerase chain reactions. U.S. Pat. Nos. 4,683,195 (issued Jul. 28, 1987 to Mullis et al) and 4,683,202 (issued to Mullis the same day) describe useful amplification and detection procedures for nucleic acids found in various biological specimens using a polymerase. Standard nucleic acid extraction techniques are mentioned by reference to Maniatis et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory, 1982), pp. 280-281. This reference is directed to a standard extraction procedure involving the use of a protease to lyse cells and phenol/chloroform extraction, the entire procedure generally taking many hours to perform and involves the use of hazardous organic solvents. It is also used to extract DNA: from hamster ovary cells by Nunberg et al, Proc. Natl. Acad. Sci. USA, 75 (11), pp. 5553-5556 (1978), from the buffy coat of whole blood specimens by Saiki et al, Bio/Technology, 3, pp. 1008-1012 (1985), and from whole blood by Bell et al, Proc. Natl. Acad. Sci. USA, 78(9), pp. 5759-5763 (1981).
For diagnostic testing to be commercially feasible, it also must be economically competitive. This means that every aspect of the procedure must be simple, easy to use and automated to some extent. Extraction of the DNA from a specimen is one aspect that requires careful development in order to obtain maximum amounts of DNA from the specimen as well as economic advantages. Moreover, in situations where a diagnostic result is needed quickly the extraction procedure should be rapid.
Thus, there has been considerable activity in developing improved DNA extraction procedures which avoid the tedious and time-consuming steps noted above and the use of organic solvents. Thus, Kogan et al, N. Eng. J. Med., 317 (16), pp. 985-990 (1987) describe the extraction of DNA to detect genetic disorders by boiling cells which have been removed from whole blood by centrifugation. The extracted DNA is then subjected to amplification.
Similarly, Saiki et al, Nature, 324, pp. 163-166 (1986) describe boiling buffered buffy coat (which includes peripheral blood mononuclear cells and granulocytes) of whole blood to amplify .beta.-globin DNA. Similar work is shown in EP-A-0 237 362 for detection of sickle cell and HLA DNA. In some cases, the cells of the buffy coat are overlaid with mineral oil prior to the heating step.
While these procedures avoid the tedious phenol/chloroform extraction described above, and appear to be rapid (done in a few minutes), they are largely useful only for extraction of DNA present in large quantities in a whole blood sample, such as HLA or .beta.-globin DNA. Where the DNA of interest is present in very small quantities, such as in the case of the presence of many infectious agents (such as viruses), further improvements in extraction from whole blood, or a buffy coat fraction, are needed for sensitive detection. Moreover, there are many interferents to polymerase activity which also need to be removed from whole blood prior to amplification.
A more recent advance in the art is described in U.S. Ser. No. 178,202 (filed Apr. 6, 1988 by Higuchi) now abandoned, whereby the defects of the tedious phenol/chloroform procedure are avoided, and which allows extraction of DNA for polymerase chain reaction. It involves the use of a composition containing a nonionic lysing detergent and a proteolytic enzyme. One of the principle advantages of this method is the shortening of the time for DNA extraction to less than two hours.
While this improvement is welcome in the art, there is a continuing need to simplify DNA extraction procedures even further, especially where the DNA is present in very small concentrations in the specimen. In particular, there is a need for a rapid and effective method of extracting HIV-I DNA from whole or a peripheral blood mononuclear fraction thereof.